Peptides having a high cysteine content

ABSTRACT

The invention relates to cysteine containing peptides of the structure XXCCXXXXXXXCXXXCXXXXXXQXXCXXXCXCXXXXXXXCXXXXXX, of the structure XXCCXXXXXXXCXXXCXXXXXXXXXCXXXCXCXXXXTXXCXXXXXX and of the structure XXCCXXXXXXXCXXXCXXXXXXXXXXCXXXCXCXXXXXXXXCXXXXXX, wherein X, independently of one another, represents any naturally occurring amino acid, as well as to nucleic acid sequences encoding said peptides, to vectors comprising said sequences, as well as to pharmaceutical compositions containing said peptides and their use as pharmaceutics, particularly for the treatment of cancers.

TECHNICAL FIELD

The present invention relates to peptides with a high cysteine content,to nucleic acid sequences encoding said peptides, to vectors comprisingsaid sequences, as well as to pharmaceutical compositions containingsaid peptides and their use as pharmaceutics.

STATE OF THE ART

A microorganism (bacterium, virus, yeast, etc.) or a transferable agent(toxin, etc.) is referred to as pathogen, which can cause a disease or asymptom-free infection of a plant, animal or human organism. In thebroader sense also insects or nematodes are referred to as pathogens,which can transfer infectious agents to organisms. Biological organismsare provided with a complex and very efficient blocking system againstattacks of pathogens. It is the principal task of this blocking andimmune system, respectively, to prevent the intrusion of the pathogensand to destroy the already invaded pathogens. Simple animals in thisregard also possess several defense agents and protection mechanisms.The immune system of vertebrates is a very complex network of cellularand humoral mechanisms with the additional ability of being able to formspecific antibodies against single pathogens and to thereby destroythem.

When the organism's own defenses are not sufficient to destroy thepathogen, additional auxiliary means have to be utilized, such as forexample antibiotics, fungicides, antiviral and other agents. However aserious problem of this chemical control of pathogens is the fact, thatthe pathogen strains very often gain resistance to the utilized means.This adaptation can be overcome temporarily with increasing doses ofactive agent, but in the long term new chemical and biological meanswill always be required for the control of pathogens.

Peptides are compounds composed of amino acids, which are referred to asdi-, tri-, oligo- or polypeptides depending on the number of thecontained amino acid units. In the case of proteins polypeptides with amolar mass greater 10 kDa are concerned. In the scope of the presentinvention the term “peptide” is used for polypeptides with a molar masssmaller than 10 kDa. Proteins and peptides, respectively, aresignificantly involved in almost all of the cellular processes. Theircentral position in the processes of life is mirrored in that thegenetic information is eventually expressed in the form of polypeptides.

On the basis of their biological tasks proteins are referred to asenzymes, transport, structure, defense, contractile, receptor proteinsetc. In other biological tasks peptides and some mostly smaller proteinsstand to the fore, such as the hormone effect (e.g. insulin, oxytocin),signal transmission to receptors (e.g. encephalins), control of theimmune defense (cytokines) or non-specific defense (e.g. defensine).Many biologically highly effective peptides are released from biggerprecursor proteins generally just at the location of action. Thus, theycan display optimal effect.

The use of peptides as a pharmaceutical is very limited despite theirhigh pharmacological effectiveness, their low toxicity and their goodbiocompatibility. A basic barrier consists in, that in the organismpeptides are decomposed mostly very rapidly and are made ineffectivethereby. It has been attempted for a long-time to prevent this rapidmetabolization through the integration of hydrolysis resistantstructural elements such as for example D-amino acids or by some otherway. The problem is, that the therapeutical effectiveness is affectedvery strongly through such structural changes.

Per orally administered proteins/peptides are decomposed in thegastrointestinal tract and thus are made virtually ineffective. Hencethis path of administration is very problematic for peptidepharmaceutics. Due to this reason the body-identic peptides such asinsulin, erythropoietin, etc., which are used in therapy, areadministered predominantly (>90%) only parenterally (throughinjections).

Another technical problem for the application of larger peptides istheir preparation at an industrial scale, which could be solvedsatisfactorily only in a few cases up to now.

Contrary to the body's own peptides the parenteral application ofbody-alien polypeptides has to overcome a basic difficulty. The immunesystem of all of the higher organisms forms specific antibodies againstsuch “non-self” peptides, which try to neutralize and to remove thealien peptide, respectively. This antigenity of alien peptides can besuppressed only artificially, e.g. by means of immunosuppressives, or bycomplicated constructs, whereby further side effects can be caused. Asthe newest example ethanercept (anti-TNF receptor protein), which isused in the therapy of rheumatoid arthritis, can be mentioned.

Body-alien polypeptides are decomposed by proteolytic enzymes in theorganism. The smaller peptide segments, which are formed thereby, elicitantigen reactions, which lead to the formation of antibodies.Polypeptides, which are stable against proteolytic enzymes, should alsoprincipally exhibit a clearly smaller antigenity. Substances, whichinhibit proteolytic enzymes effectively, can be used in the therapy ofseveral pathologies. Such protease inhibitors were already utilized forthe treatment of the HIV infection.

Thionins are small, extremely basic peptides of plant origin, which arecharacterized in an unusually high content of cysteine. The thionins aremostly composed of 45 to 48 amino acid units (Römpp Lexikon der Chemie9. Edition, Thieme Verlag Stuttgart 1992). They contain mostly 6 or 8cysteine groups, which form 3 or 4 intramolecular disulfide bridges(—S—S—) accordingly and thereby additionally stabilize the structure ofthe peptide. Due to their relatively low (about 5 kDa) molecular weightand their exceptionally stable structure it is to be expected thatthionins feature more pharmacologically specific properties, whichhowever were recognized and used only to a minor extent up to now.

In the technical literature only about 50 single thionins have beendescribed up to now. The most known thionins are viscotoxins frommistletoe (Viscum album), hordothionins from barley (Hordeum vulgaris),purothionins from wheat (Triticum sativum), avenothionins from oat(Avena-sativa), pyrulariathionins from buffalo nut (Pyrularia-pubera)and crambin from Crambus abessinicus (D. E. A. Florack and W. J.Stiekema, Plant. Mol. Biol. 26, 25-37 (1994)).

The exact physiologic effect of the plant thionins could not beclarified clearly up to now. It was observed in the case of some thioninproducing plants, that in the case of infections with bacteria or fungithese react with an increased expression of their thionins. It wasassumed for this reason, that thionins could fulfill some kind offunction as a defense agent. Consequently the integration of thioningenes from oat (Avena sativa) into other, thionin-free economic plantswas carried out with the intention to breed novel types of economicplants (e.g. rice or potato) with an increased resistance against fungiand bacteria (U.S. Pat. No. 5,942,663; U.S. Pat. No. 6,187,995).

Through the integration of the alpha-thionin gene sequences from barleyinto the genome of the tobacco plant e.g. a tobacco type was createdwith a resistance against Pseudomonas syringae (M. J. Carmona, A.Molina, J. A. Lopez-Fando and F. Garcia-Olmedo, Plant J. 3, 457-462(1993)). The inhibition of Phytophora infestans on potato leaf bythionins was also described (A. Molina, P. A. Groy, A. Fraile, R.Sanchez-Monge and F. Garcia-Olmedo, Plant Science 92, 672-679 (1993)).

The integration of thionin producing genes opens the way for thebreeding of a large variety of novel plant types with a clearlyincreased resistance against pests. Thereby the use of pesticides, whichintensely pollute humans and the environment, could clearly be reduced.The essential precondition for this type of use, besides the higheffectiveness of the utilized thionins, is their preferably low toxicityfor animals and humans. The effectiveness of a particular thionin ismost efficient only against particular pests and pathogens,respectively, such that new thionin structures have to be found for thebattle against further pathogens.

Thionins are very often referred to as generally toxic for animals,insects, bacteria, yeast and mammal cells (D. E. A. Florack and W. J.Stiekema, Plant. Mol. Biol. 26, 25-37 (1994)). However single thioninsfeature a toxicity differing very strongly from one another, which isdependent on both the structure of the thionins and the precise methodof administration. Viscotoxin B for example shows a low toxicity,although its structure only differs at 4 amino acid positions from thesequence of the clearly more toxic viscotoxin A-1. The toxic effect ofdifferent thionins is explained in general by the permeabilization ofthe cytosolic membrane for alkali ions and the necrosis of the cellscaused thereby. The mechanism of action on cellular level wasinvestigated in some cases in detail such as e.g. the formation ofmembrane ion channels by Pyrularia thionins (Hughes P. et al J. Biol.Chem. (2000) 275, 823-827). However it was shown in a study carried outrecently, that viscotoxins are toxic for human lymphocytes, butpurothionins from wheat do not exert a toxic effect on the samelymphocytes (Büssing A. et al. Eur. J. Biochem. (1999) 262, 79-87).

Considerable amounts of thionins are present in the most corn types usedfor nutrition, they are also contained in numerous feeding stuffs andcomestible goods. The per oral toxicity of these thionins was found tobe mostly not significant for humans and animals, probably because ofthe decomposition of the thionins in the gastrointestinal tract andbecause of the thermal decomposition in the oven.

A direct human therapeutic use of a thionin as pure substance or of astandardized mixture of pure thionins has not been described in thetechnical literature up to now.

It is known that some preparations from mistletoe (Viscum album), usedfor a long time in adjuvant cancer therapy, contain smaller amounts ofthionins which are referred to as viscotoxins. However, regarding theoncological effectiveness of these viscotoxins however there is noreliable knowledge present. Several authors even suggest explicitly,that the viscotoxins do not possess relevance for the cancer therapeuticeffectiveness of the mistletoe preparations (U.S. Pat. No. 5,547,674).

The commercially available mistletoe preparations are for this reasonnot standardized on their viscotoxin content. Their actual viscotoxinconcentration is variable, but lies in all of the cases under the limitof 1 μg/ml. From the normal dose of 2-4 ml mistletoe extract injectionsolution thus a patient receives 0.004 mg viscotoxin per day at maximum.With the determined toxicity of the viscotoxins of LD₅₀=0.1-1 mg/kg inthe case of mammals, in fact no relevant cytotoxic effect can occur inthe case of a mature human (70 kg body weight), which could lead to thekilling of tumour cells.

In the technical literature the oncological effectiveness of themistletoe preparations is predominantly accredited to the lectins (sugarbinding proteins) contained in the extract (DE 4 221 836). Thesemistletoe lectins show an immunostimulating effect in low dosages (1ng/kg), which was confirmed in vitro several times (EP-A-0 602 686). Asa result several commercially available mistletoe preparations arestandardized on their content of mistletoe lectins. Nonetheless the invivo cancer therapeutic effectiveness of pure isolated mistletoe lectinscould not be confirmed. Hence several authors assume a contribution frommistletoe polysaccharides or from further components of the mistletoeextracts not identified up to now (J. R. Ochocka and A. Piotrowski, Can.J. Plant Pathol. 24, 21-28 (2002)).

Latterly it was proposed to utilize the immunostimulating effect of thePyrularia thionin observed at lower concentrations for the control oftumors (WO 02/22159). WO 02/22159 describes also the use of the thionintogether with the strongly stimulating IL-2. In this context it is aconcern, that a strong but non-specific immunostimulation also enhancesthe growth of the cancer cells, according to experience even more sothan the growth of normal cells.

Thionins, which are clearly more toxic against cancer cells than againstnormal cells, would be very suitable for the therapy of cancer. Howeversuch thionins could not be identified up to now. It was proposed (WO96/41608) to bind a Pyrularia thionin to particular CD5+ antibodies,which are specifically targeted against cancer cells. This complexshould be then used for the selective control of cancer cells.

Cancer is not a uniform disease but a generic term for more than 200different forms of malignant diseases. Almost every tissue can causecancer-like degenerations, sometimes even several different types.Despite of this dissimilitude obviously all tumors are generated throughthe degeneration of similar basic processes.

The established therapies for cancer try to controvert the basiccharacteristics of the disease, namely growth, invasion andmetastization. For the treatment of cancer chemotherapeutics are veryimportant means which are utilized besides surgery and radiation.Particular malignant tumors (testicular cancer) can be treated withcytostatics successfully. Unfortunately the most frequent malignanttumors (cancer of the breast, lungs and prostate as well as colon) cannot be cured by means of the chemotherapy. If the development of thetumor can not be stopped the cancer cells become more and moreaggressive and start to spread themselves after a particular phase(metastasis) and to damage vitally important organs until inoperability.Thereby they lead to the death of the patients.

A basic disadvantage of the cytotoxic cancer therapeutics actingnon-selectively lies in that they also kill numerous healthy cells.Hence severe side effects have to be taken into account in the case oftheir use. Another disadvantage of the actual chemotherapies is that thetumour cells are developing resistances against the utilized cytostaticsduring the treatment. This requires the constant increasing of theapplied dose rate, with which an increase of the toxic side effects issimultaneously connected, or the use of a mixture of severalcytostatics.

AIDS is a complex disease syndrome with chronic damaging of the T4lymphocytes and further immune cells, which forms due to an infectionwith the HIV retroviruses. The common immune deficiency referred to asAIDS establishes only after the immune system has lost the struggleagainst the infection. In the AIDS phase the patient does not have animmune defense against different, mostly minor, infections and dies asthe consequence of such diseases.

During the search of factors which participate in the regulation of thecellular and humoral immune processes in addition to polypeptides, thenovel class of the carbon suboxide derivatives was recently discovered(DE 196 00 301, EP 0874 851). These natural compounds derived frominorganic carbon suboxide C₃O₂ feature inter alia a surprisinglyspecific interaction with certain immunologically important proteinssuch as immunoglobulins (Ig) or F_(c) receptors. Further it wasascertained, that these carbon suboxide derivatives are capable ofinfluencing significantly the pharmacological-toxicological propertiesof the peptides described herein but also of other peptides having highcysteine content.

DESCRIPTION OF THE INVENTION

Objective of the present invention is to provide peptides, which supportthe natural defenses of herbal, animal or human organisms againstbacterial, fungal, viral or other pathogens. This objective is solvedaccording to invention by the cysteine containing peptides according toindependent claim 1, by the nucleic acid sequence according toindependent claim 14, by the derivatives according to independent claim17, by the DNA vector according to independent claim 18, by themonoclonal antibodies according to independent claim 19, by thepharmaceutical composition according to independent claim 28, by thepharmaceutics according to independent claim 34 and by the methodsaccording to independent claims 36, 38 and 40. Further advantageousaspects, details and designs of the invention result from the dependentclaims, the description and the drawings.

The configuration of the peptides according to invention is illustratedin the one-letter code of the amino acids as follows, namely: A:alanine, C: cysteine, D: aspartic acid, E: glutamic acid, F:phenylalanine, G: glycine, H: histidine, I: isoleucine, K: lysine, L:leucine, M: methionine, N: asparagine, P: proline, Q: glutamine, R:arginine, S: serine, T: threonine, V: valine, W: tryptophan and Y:tyrosine.

The secondary, tertiary and quaternary structure of peptides issignificantly defined by the type of the side chains of the amino acidsfrom which the respective peptide is constructed. In this context thenaturally occurring amino acids can be divided into groups, where thetype of the side chain represents the classification criteria. Aminoacids with aliphatic side chains are glycine, alanine, valine, leucineand isoleucine. Amino acids with aliphatic side chains with hydroxylgroup are serine and threonine. Amino acids with aromatic side chainsare phenylalanine, tyrosine and tryptophan. Amino acids with basic sidechains are lysine, arginine and histidine. Amino acids with acid sidechains are aspartate and glutamate. Amino acids with amide side chainsare asparagine and glutamine. Amino acids with sulphur containing sidechains are cysteine and methionine. The amino acid proline has asecondary amino group and can not be divided into one of the mentionedclasses. A special significance for the influencing of the threedimensional structure and hence also an influencing of the biologicaleffectiveness can especially result through the formation of the esterderivatives of the amino acids containing hydroxyl groups. The esterformation can be conducted preferably with phosphoric acid. A suchlikederivatization can be carried out chemically-synthetically orenzymatically by means of so called phospho-kinases.

The present invention refers to cysteine containing peptides of thestructure XXCCXXXXXXXCXXXCXXXXXXQXXCXXXCXCXXXXXXXCXXXXXX, of thestructure XXCCXXXXXXXCXXXCXXXXXXXXXCXXXCXCXXXXTXXCXXXXXX and of thestructure XXCCXXXXXXXCXXXCXXXXXXXXXXCXXXCXCXXXXXXXXCXXXXXX, wherein Xrepresents a replacement character. All of the X can be chosenindependently from one another and mean any of the above mentionednaturally occurring amino acids.

As naturally occurring amino acids are included in addition to theestablished L-amino acids also D-amino acids as well as amino acidderivatives such as for example 4-hydroxyproline, N,N,N-trimethyllysine,3-methylhistidine, 5-hydroxylysine, O-phosphoserine, γ-carboxyglutamate,ε-N-acetyllysine, ω-N-methylarginine, citrulline or ornithine.

The disulphide bridges are formed preferably between the cysteines shownin the following, such that the following preferred structures areresulting:

Especially preferred embodiments of the present invention refer tocysteine containing peptides of the mentioned general structures,whereas further amino acids are defined at certain positions of thepeptide. Preferred are peptides, wherein at position 1 the amino acid Kand/or at position 2 the amino acid S and/or at position 5 the aminoacid R and/or at position 6 the amino acid N and/or at position 7 theamino acid T and/or at position 8 the amino acid L and/or at position 10the amino acid R and/or at position 11 the amino acid N and/or atposition 13 the amino acid Y and/or at position 17 the amino acid Rand/or at position 20 the amino acid G is located.

Further preferred are the following peptides of the structure 1 and ofthe structure XXCCXXXXXXXCXXXCXXXXXXQXXCXXXCXCXXXXXXXCXXXXXX,respectively, wherein at position 15 the amino acid G and/or at position19 the amino acid T and/or at position 27 the amino acid Q and/or atposition 28 the amino acid R and/or at position 31 the amino acid Dand/or at position 33 the amino acid I and/or at position 34 the aminoacid H and/or at position 35 the amino acid V and/or at position 36 theamino acid T and/or at position 37 the amino acid T and/or at position38 the amino acid T and/or at position 43 the amino acid S and/or atposition 44 the amino acid H and/or at position 46 the amino acid S islocated.

Further preferred are the following peptides of the structure 2 and ofthe structure XXCCXXXXXXXCXXXCXXXXXXXXXCXXXCXCXXXXTXXCXXXXXX,respectively, wherein at position 15 the amino acid G and/or at position19 the amino acid T and/or at position 23 the amino acid Q and/or atposition 27 the amino acid Q and/or at position 28 the amino acid Rand/or at position 31 the amino acid D and/or at position 33 the aminoacid I and/or at position 34 the amino acid H and/or at position 35 theamino acid V and/or at position 36 the amino acid T and/or at position38 the amino acid T and/or at position 43 the amino acid S and/or atposition 44 the amino acid H and/or at position 46 the amino acid S islocated.

Further preferred are the following peptides of the structure 3 and ofthe structure XXCCXXXXXXXCXXXCXXXXXXXXXCXXXCXCXXXXXXXXCXXXXXX,respectively, wherein the following amino acid combinations areresiding: Position AS Position AS Position AS 15 G and 19 T or 23 Q or28 G or 29 R or 32 D or 34 I or 35 H or 36 V or 37 T or 38 T or 39 T or44 S or 45 H or 47 S 19 T and 15 G or 23 Q or 28 G or 29 R or 32 D or 34I or 35 H or 36 V or 37 T or 38 T or 39 T or 44 S or 45 H or 47 S 20 Tand 15 G or 23 Q or 28 G or 29 R or 32 D or 34 I or 35 H or 36 V or 37 Tor 38 T or 39 T or 44 S or 45 H or 47 S 23 Q and 15 G or 19 T or 28 G or29 R or 32 D or 34 I or 35 H or 36 V or 37 T or 38 T or 39 T or 44 S or45 H or 47 S 24 Q and 15 G or 19 T or 28 G or 29 R or 32 D or 34 I or 35H or 36 V or 37 T or 38 T or 39 T or 44 S or 45 H or 47 S 28 Q and 15 Gor 19 T or 23 Q or 29 R or 32 D or 34 I or 35 H or 36 V or 37 T or 38 Tor 39 T or 44 S or 45 H or 47 S 29 R and 15 G or 19 T or 23 Q or 29 R or32 D or 34 I or 35 H or 36 V or 37 T or 38 T or 39 T or 44 S or 45 H or47 S 32 D and 15 G or 19 T or 23 Q or 28 Q or 29 R or 34 I or 35 H or 36V or 37 T or 38 T or 39 T or 44 S or 45 H or 47 S 34 I and 15 G or 19 Tor 23 Q or 28 Q or 29 R or 32 D or 35 H or 36 V or 37 T or 38 T or 39 Tor 44 S or 45 H or 47 S 35 H and 15 G or 19 T or 23 Q or 28 Q or 29 R or32 D or 34 I or 36 V or 37 T or 38 T or 39 T or 44 S or 45 H or 47 S 36V and 15 G or 19 T or 23 Q or 28 Q or 29 R or 32 D or 34 I or 35 H or 37T or 38 T or 39 T or 44 S or 45 H or 47 S 37 T and 15 G or 19 T or 23 Qor 28 Q or 29 R or 32 D or 34 I or 35 H or 36 V or 38 T or 39 T or 44 Sor 45 H or 47 S 38 T and 15 G or 19 T or 23 Q or 28 Q or 29 R or 32 D or34 I or 35 H or 36 V or 37 T or 39 T or 44 S or 45 H or 47 S 39 T and 15G or 19 T or 23 Q or 28 Q or 29 R or 32 D or 34 I or 35 H or 36 V or 37T or 38 T or 44 S or 45 H or 47 S 44 S and 15 G or 19 T or 23 Q or 28 Qor 29 R or 32 D or 34 I or 35 H or 36 V or 37 T or 38 T or 39 T or 45 Hor 47 S 45 H and 15 G or 19 T or 23 Q or 28 Q or 29 R or 32 D or 34 I or35 H or 36 V or 37 T or 38 T or 39 T or 44 S or 47 S 47 S and 15 G or 19T or 23 Q or 28 Q or 29 R or 32 D or 34 I or 35 H or 36 V or 37 T or 38T or 39 T or 44 S or 45 H

Further preferred are the following peptides of the structure 4 and ofthe structure XXCCXXXXXXXCXXXCXXXXXXXXXCXXXCXCXXXXXXXCXXXXXX,respectively, wherein the following amino acid combinations areresiding: Position AS Position AS Position AS 15 G and 19 T or 27 Q or28 R or 31 D or 33 I or 34 H or 35 V or 36 T or 37 T or 38 T or 43 S or44 H or 46 S 19 T and 15 G or 27 Q or 28 R or 31 D or 33 I or 34 H or 35V or 36 T or 37 T or 38 T or 43 S or 44 H or 46 S 27 Q and 15 G or 19 Tor 28 R or 31 D or 33 I or 34 H or 35 V or 36 T or 37 T or 38 T or 43 Sor 44 H or 46 S 28 R and 15 G or 19 T or 27 Q or 31 D or 33 I or 34 H or35 V or 36 T or 37 T or 38 T or 43 S or 44 H or 46 S 31 D and 15 G or 19T or 27 Q or 28 R or 33 I or 34 H or 35 V or 36 T or 37 T or 38 T or 43S or 44 H or 46 S 33 I and 15 G or 19 T or 27 Q or 28 R or 31 D or 34 Hor 35 V or 36 T or 37 T or 38 T or 43 S or 44 H or 46 S 34 H and 15 G or19 T or 27 Q or 28 R or 31 D or 33 I or 35 V or 36 T or 37 T or 38 T or43 S or 44 H or 46 S 35 V and 15 G or 19 T or 27 Q or 28 R or 31 D or 33I or 34 H or 36 T or 37 T or 38 T or 43 S or 44 H or 46 S 36 T and 15 Gor 19 T or 27 Q or 28 R or 31 D or 33 I or 34 H or 35 V or 37 T or 38 Tor 43 S or 44 H or 46 S 38 T and 15 G or 19 T or 27 Q or 28 R or 31 D or33 I or 34 H or 35 V or 36 T or 37 T or 43 S or 44 H or 46 S 43 S and 15G or 19 T or 27 Q or 28 R or 31 D or 33 I or 34 H or 35 V or 36 T or 37T or 38 T or 44 H or 46 S 44 H and 15 G or 19 T or 27 Q or 28 R or 31 Dor 33 I or 34 H or 35 V or 36 T or 37 T or 38 T or 43 S or 46 S 46 S and15 G or 19 T or 27 Q or 28 R or 31 D or 33 I or 34 H or 35 V or 36 T or37 T or 38 T or 43 S or 44 HThe abbreviation AS represents amino acid.

The Abbreviation AS represents amino acid.

Very especially preferred are cysteine containing peptides of thestructure

KSCCRNTLGRNCYNGCRFTGGSQPTCGRLCDCIHVTTTTCPSSHPS (hellethionin-A),

KSCCRNTLGRNCYNACRFTGGSQPTCGRLCDCIHVTTTTCPSSHPS (hellethionin-B1)

KSCCRNTLARNCYNACRFTGGSQPTCGRLCDCIHVTTTTCPSSHPS (hellethionin-B2),

KSCCRNTLGRNCYNACRLPGTPQPTCATLCDCIHVTTPTCPSSHPR (hellethionin-B3),

KSCCRNTLARNCYNACRFTGTSQPYCARLCDCIHVTTPTCPSSHPR (hellethionin-B4),

KSCCRNTLARNCYNACRFTGGSQPTCATLCDCIHVTTPTCPSSHPR (hellethionin-B5),

KSCCRNTLARNCYNVCRFGGGSQAYCARFCDCIHVTTSTCPSSHPS (hellethionin-B6),

KSCCRNTLGRNCYNACRLTGTSQATCATLCDCIHVTATTCRPPYPS (hellethionin-C),

KSCCRNTLARNCYNACRFTGGSQPTCGILCDCIHVTTTTCPSSHPS (hellethionin-D),

KSCCRNTLGRNCYAACRLTGLFSQEQCARLCDCITVTTPTPCPRTHPS (hellethionin-E1) and

KSCCRNTLGRNCYAACRLTGTFSQEQCARLCDCITVTTPTPCPRTHPS (hellethionin-E2).

Especially preferred are further derivatives of the afore-mentionedhellethionins, i.e. derivatives of hellethionin-A, hellethionin-B1,hellethionin-B2, hellethionin-B3, hellethionin-B4, hellethionin-B5,hellethionin-B6, hellethionin-C, hellethionin-D and hellethionin-E1,wherein the following amino acids (initial AS) have been replaced by theamino acids (substitution AS) listed on the side: Initial ASSubstitution AS Ala Ser Arg Lys Asn Gln or His Asp Glu Cys Ser Gln AsnGlu Asp Gly Pro His Asn or Gln Ile Leu or Val Leu Ile or Val Lys Arg orGln or Glu Met Leu or Ile Phe Met or Leu or Tyr Ser Thr Thr Ser Trp TyrTyr Trp or Phe Val Ile or Leu

Further also the use of peptidomimetics is possible. Herein chemicalsubstances are concerned, which imitate one of the afore-mentionedpeptides.

The present invention also comprises ester derivatives, amidederivatives, salt derivatives, cyclic derivatives and derivatives with amodified backbone of the mentioned peptides.

Amino acids with carboxylate groups can be transferred for example intoa salt, or into an ester, preferred into a C₁-C₁₆ ester or an amide,optionally with one or two alkyl moieties, preferred with C₁-C₁₆ alkylmoieties. Hydroxyl groups of for example tyrosine or serine can betransferred into an ester or ether. Further also the formation ofacetals, ketals or carbonates is possible. Such moieties preferablyconsist of 1 to 16 carbon atoms. Further all of the other known OHprotecting groups can be utilized. The alkyl moieties used in theprotecting groups can bear further substituents for example halogens,amino groups, hydroxyl groups, carbonyl groups, thiol groups, arylgroups, alkyl branchings, carboxyl groups, nitro groups, amide groupsand/or ester groups.

Very especially preferred in the scope of the present invention are thecysteine containing peptide compounds hellethionin-A, hellethionin-B1,hellethionin-B2, hellethionin-B3, hellethionin-B4, hellethionin-B5,hellethionin-B6, hellethionin-C, hellethionin-D, hellethionin-E1 andhellethionin-E2 as well as derivatives of these cysteine containingpeptide compounds with amino acid variations at up to 15 positions ofthe cysteine containing peptide compound respectively at up to 15positions of the derivatives of these cysteine containing peptidecompounds, whereas the amino acid cysteine is not subject to variationregarding position and type. It is especially preferred, if the aminoacid variations occur at up to 13 positions, at up to 11 positions, atup to 9 positions, at up to 7 positions, at up to 5 positions, at up to4 positions, at up to 3 positions, at up to 2 positions and only at oneposition, respectively, of the peptide compounds and of the derivativesof the peptide compounds, respectively.

It is especially preferred, if the cysteine containing peptidecompounds, hellethionin-A, hellethionin-B1, hellethionin-B2,hellethionin-B3, hellethionin-B4, hellethionin-B5, hellethionin-B6,hellethionin-C, hellethionin-D, hellethionin-E1 and hellethionin-E2 aswell as derivatives of these cysteine containing peptide compoundsfeature one of the above-mentioned amounts of amino acid variations andif simultaneously at position 1 the amino acid K and/or at position 2the amino acid S and/or at position 5 the amino acid R and/or atposition 6 the amino acid N and/or at position 7 the amino acid T and/orat position 8 the amino acid L and/or at position 10 the amino acid Rand/or at position 11 the amino acid N and/or at position 13 the aminoacid Y and/or at position 17 the amino acid R and/or at position 20 theamino acid G is located.

Especially preferred embodiments of the present invention refer tocysteine containing peptides of the mentioned general structures,wherein amino acids are located at particular positions of the peptide,which are selected from a particular group of amino acids defined above.Preferred are peptides, wherein at position 1 an amino acid with basicside chain and/or at position 2 an amino acid with aliphatic side chainwith hydroxyl group and/or at position 5 an amino acid with basic sidechain and/or at position 6 an amino acid with amide side chain and/or atposition 7 an amino acid with aliphatic side chain with hydroxyl groupand/or at position 8 an amino acid with aliphatic side chain and/or atposition 10 an amino acid with basic side chain and/or at position 11 anamino acid with amide side chain and/or at position 13 an amino acidwith aromatic side chain and/or at position 17 an amino acid with basicside chain and/or at position 20 an amino acid with aliphatic side chainis located.

Of course the present invention comprises also peptides of the mentionedgeneral structures, which are bearing functional modifications at one orat both of their ends. Especially comprised are also peptides havinglonger chains, i.e. peptides, the amino acid chain of which exceeds thementioned general structures. Thus the peptides according to theinvention represent in these cases only a section of a greater peptide.The functionality of the peptides according to the invention, and sotheir effectivity, may not be influenced significantly by thesefunctional modifications.

From the structures 1-4 according to the invention as well as theformulas XXCCXXXXXXXCXXXCXXXXXXQXXCXXXCXCXXXXXXXCXXXXXX,XXCCXXXXXXXCXXXCXXXXXXXXXCXXXCXCXXXXTXXCXXXXXX andXXCCXXXXXXXCXXXCXXXXXXXXXXCXXXCXCXXXXXXXXCXXXXXX according to theinvention also peptides are comprised, which are bearing before the X atposition 1 and/or after the X at position 46 and 47, respectively,arbitrary end groups or featuring still another oligopeptide chain withup to 50 amino acids, preferred with up to 30 amino acids, morepreferred with up to 15 amino acids and especially preferred with up to7 amino acids.

In addition the present invention refers to the nucleic acid sequences,which are encoding for the mentioned cysteine containing peptides, andalso to the corresponding RNA sequences and the corresponding DNAsequences as well as the corresponding anti-sense DNA and thecorresponding anti-sense RNA. The present invention comprises also DNAvectors and DNA constructs, which contain a cDNA or DNA corresponding toa peptide according to the invention as well as a suitable promoter anda suitable enhancer if necessary.

In addition also monoclonal antibodies are comprised, which are targetedagainst an epitope of the mentioned cysteine containing peptides.

The peptides having a high cysteine content according to the inventionare composed of 46 and 48 amino acids, respectively. The count of thepositions is carried out starting from the free NH₂-end of the aminoacid chain.

From the above-mentioned hellethionins the peptides referred to withhellethionin-A (HT-A), hellethionin-C (HT-C) and with hellethionin-D(HT-D) were isolated individually by the methods described in thefollowing. In the case of hellethionin-B and hellethionin-E a mixture ofseveral isoforms is present, whereas the amino acid sequence of sixisoforms (HT-B1, HT-B2, HT-B3, HT-B4, HT-B5, HT-B6) and of two isoforms(HT-E1 and HT-E2), respectively, was determined. The peptides accordingto the invention can be used as pure substances, as a mixture ofisoforms or as a mixture of isoforms together with one or more otherhellethionins, preferably as a mixture of several hellethionins.

The amino acid sequence of the isolated or the synthetically producedpeptides according to the invention was determined by successivedegradation of the peptide chain according to the Edman method andsubsequent HPLC identification of the PTH (phenylthiohydantoin)derivatives. The enzymatic fragmentation of the peptides usual in thecase of this method could not be carried out directly due to theirincreased enzymatic stability. For this reason initially a reduction andopening, respectively, of the disulphide bridges carried out by means ofvinylpyridine was required.

Another confirmation of the amino acid composition of the peptidesaccording to the invention was carried out by the determination of theirmolar masses. For this purpose mass spectrometry with ESI(electro-spray-ionization) and the MALDI(matrix-assisted-laser-desorption-ionization) technique was utilized.

The three-dimensional structure of the peptide HT-D determined by NMRspectroscopy is illustrated in FIG. 1. The total structure is similar tothe shape of a capital Greek gamma (Γ). The long arm in this structureanalogy is formed through the two helices running contrarily, whereasthe short arm consists of the short beta-sheet. The both helices areconnected through a loop between the amino acids in the positions 17 and24.

A special contribution to the stability of the peptides according to theinvention is guaranteed through the total of four disulphide bridgesbetween the cysteine units in the positions 3-40 (or respectively 42),4-32, 12-30 (or respectively 31) and 16-26 (or respectively 27). Theextraordinary stability obtained thereby contrary to proteolytic andother enzymes is a specific property with important meaning for the useof the peptides described herein.

The preferred peptides in the scope of the present invention arecharacterized in an astonishing high conservation of their amino acidsequences. This conservation pertains to up to 36 positions, thus about% of the total chain. The variations in the composition of thepolypeptide chain are mostly limited to 15 or even less positions.

Especially constant is the position of the cysteine moieties, which arelocated in the peptides according to the invention at the positions 3,4, 12, 16, 26 or respectively 27, 30 or respectively 31, 32 and 40 orrespectively 42. In the sequences referred to with HT-E1 and HT-E2 thelast three cysteine units are displaced each by one (27 instead of 26)(31 instead of 30) or by two positions (42 instead of 40). The cysteineunits are connected through disulphide bridges in pairs. From thenumerous possible combinations of cysteine units in the peptidesaccording to invention the connections 3→40 (or respectively 42), 4→32,12→30 (or respectively 31) and 16→26 (or respectively 27) are preferablyformed, thereby defining a particular secondary structure of thepeptides according to the invention.

The use of the peptides according to the invention provides severaladvantages in comparison to the thionins with 3 disulphide bridges knownup to now. Firstly due to the presence of the additional cysteine bridgethe peptides according to invention possess an improved stability incomparison to proteolytic enzymes and show accordingly a lowerimmunogenity than for example the already mentioned viscotoxins. Anotheradvantage of the peptides according to the invention lies in theirsurprisingly good solubility in water and in the better bioavailabilityachieved thereby. This advantage becomes especially clear in thecomparison with the thionins with 4 disulphide bridges known up to now.The previously described thionin peptides with 4 disulphide bridges suchas for example purothionins, avenothionins or hordothionins are stronglybasic and very lipophilic and therefore only poorly soluble in water.They are extracted from the corresponding grain seeds by means of apolarsolvents such as petrol ether. Hence aqueous solutions of these peptidesare difficult to produced and are difficult to use.

Isolation and Preparation of the Peptides According to Invention

The present invention comprises also methods for the extraction of thecysteine containing peptides according to the invention. Such a methodaccording to invention is represented by the extraction of Helleborusplant species. Especially preferred in this case is defatting of theplant material using non-polar solvents or mixtures of non-polarsolvent, particularly using tert.-butylmethylether, carried out as thefirst step of the method. The isolation of the peptides having a highcysteine content according to the invention or of mixtures of thepeptides according to the invention is carried out from plants of thefamily Ranunculaceae (buttercup family) and preferably from plants ofthe genus Helleborus (Christmas rose). Air dried plant material,preferably the subterrestrial parts of the plant (roots and rootstock)are used for the isolation. Initially the preferred defatting of theplant material is carried out with a non-polar solvent, preferably TBM(ter.-butylmethylether). The defatted and air dried roots aresubsequently extracted with mixtures of organic alcohol/acid, preferablymethanol/formic acid, or with solvent mixtures containing water,preferably water/ethanol, respectively with diluted acids, preferablyacetic acid 0.1-12%.

The filtered extraction solution is reduced under vacuum up to 1/10 ofthe initial volume and then treated with an adsorption material,preferably active carbon. The filtrate is reduced under vacuum and thedry residue is resolved in a minimal amount of water. The pH value ofthe aqueous solution is adjusted in the acid region, preferably pH0.1-3.0, by adding diluted hydrochloric acid. The aqueous solution ismerged with a multiple, preferably tenfold, volume of a cooled organicsolvent or solvent mixture, preferably ethanol/acetone 1:3. Thelight-yellow precipitate that is formed is filtered off, vacuum driedand used for the further purification procedure.

An alternative method for the extraction of the peptides according tothe invention is carried out by their selective coupling to ionexchanger resins, preferably to slightly acidic ion exchangers. Thepeptides coupled to the resin are released by means of a treatment withstrongly ionic solutions, preferably HCl or NaCl.

The isolation of the peptides according to the invention can also becarried out by selective coupling to special solid phases, preferred tosolid phases, which enable an especially selective isolation throughantigen-antibody or similar highly specific interactions.

Another method for the extraction of the peptides according to theinvention is carried out by the separation of their native mixtures bymeans of high pressure liquid chromatography (HPLC) on a preparativescale. Mixtures of acetonitrile/water with linear gradient are used aseluents. A pH value between 1 and 1.5 is adjusted by adding a strongacid, preferably trifluoroacetic acid.

In addition the present invention relates also to methods for syntheticgeneration of the cysteine containing peptides according to theinvention and to functional derivatives of these peptides by peptidesynthesis. The synthetic generation of the peptides according to theinvention is carried out through stepwise coupling of the single aminoacid building blocks. Therefore automatic methods of peptide synthesisare utilized, wherein methods of solid phase and methods of liquid phaseare preferred. The methods are described in detail in M. Bodanszky,Principles of Peptide Synthesis (B. M. Trost Edit.) Springer Verlag1994.

In addition the present invention comprises the generation of thecysteine containing peptides according to the invention by methods ofgene technology. Preferred is gene technological generation through theintegration of genes of the peptides according to the invention into thegenetic material of a plant, preferred grain types having a high proteinoutput. An especially preferred embodiment in this context is thereplacement of the thionin genes of a thionin producing grain with thethionin genes of species of the plant Helleborus.

The generation of the peptides according to the invention is alsopossible through the transgenic expression of the cDNA sequences. Theidentified DNA segments (as cDNA clones, as genome DNA clones or throughDNA segments prepared by oligonucleotide synthesis) can be expressed ina biological system. Preferred biological systems for the expression ofthe cysteine containing peptides according to the invention are culturesof certain easily available microorganisms such as Escherichia coli,Pseudomonas or yeast.

Cell cultures of plants, into which the gene sequences encoding for thepeptides according to invention are integrated, also provide goodresults for the generation of the peptides according to the invention.Known methods are used for the transformation of the utilised plantcells. Preferably used are Ti-plasmids of Agrobacterium, electroporationor micro injection. The genetically altered plant cells can be alsoregenerated as plants, in which the gene sequences encoding for thepeptides according to invention remain stably integrated in the genome.

Special chemical modifications of the peptides can be carried out, bywhich an increase of the bioavailability is achieved, for the use of thepeptides according to the invention in the pharmaceutical field. Hencethe invention also relates to the chemical modification of the peptidesaccording to the invention through their transformation into amacrocyclic compound, preferably by closing an additional bond betweenthe two amino acids at the beginning and at the end or in the vicinityof both ends of the peptide chain. The synthesized macrocyclic peptidefeatures advantageous physiological properties, especially an increasedstability against proteolytic and other enzymes. For the preparation ofthe cyclic compounds established reagents are used such ascarbodiimides, especially 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimid(EDAC).

In the scope of the present invention the hellethionins can be alsomodified through the targeted replacement of one or more amino acidunits with a natural or synthetic amino acid, preferably with anaromatic amino acid such as tyrosine. Such a modification of the aminoacid sequence can be carried out by targeted mutations of the genesegment expressing a DNA sequence of the peptide according to theinvention. Thereby preferably used is the SPI (selective pressureincorporation) method (C. Minks et al. in Tetrahedron, 56 (2000)9431-9442).

A derivative, which can be generated through minor chemical conversionof one or more amino acid building blocks of the hellethionins, is alsoa part of the present invention. Such a derivatization leads to atargeted change of the therapeutical properties of the peptides.Preferred is the conversion of the amino acids containing hydroxylgroups such as threonine or tyrosine into ester derivatives or halogenderivatives. The conversion of the amino acids containing freeCOOH-moieties into ester derivatives or amide derivatives is also a partof the present invention. The alkylation, preferably methylation, of thefree amino groups or of other functional groups leads to a betterbioavailability and is also a part of the present invention.

Use of the Peptides

The cysteine containing peptides according to the invention, mixtures ofthese cysteine containing peptides and functional derivatives of thesepeptides can be used in the treatment of diseases, especially in thetreatment of diseases caused by pathogens.

In addition the cysteine containing peptides according to the invention,mixtures of these cysteine containing peptides and functionalderivatives of these peptides can be used in the treatment of diseasescaused by bacteria, fungi or viruses.

The cysteine containing peptides according to the invention, mixtures ofthese cysteine containing peptides and functional derivatives of thesepeptides can be especially used in the treatment of diseases of humansand animals, especially in the case of large animals, preferably horses.

It has especial advantages to use the cysteine containing peptidesaccording to the invention, mixtures of these cysteine containingpeptides and functional derivatives of these peptides in the treatmentof diseases, which are caused by defective bioregulation of the immunesystem or are accompanied by a defective bioregulation of the immunesystem.

In addition the cysteine containing peptides according to the invention,mixtures of these cysteine containing peptides and functionalderivatives of these peptides can be used especially successfully in thetreatment of autoimmune diseases, in the treatment of cancer and in thetreatment of AIDS.

In addition the present invention comprises pharmaceutical compositions,which contain one or more cysteine containing peptides according to theinvention and/or functional derivatives of these peptides. Especiallypreferred are pharmaceutical compositions, which comprise in addition atleast one carbon suboxide derivative.

In the scope of the present invention under the term “carbon suboxidederivatives” natural substances are understood, which are derived frominorganic carbon suboxide C₃O₂, as they are described in DE 196 00 301,EP 0 874 851 B1 and Kerek et al., Biochim. Biophys. Acta 1567, 213-220(2002). In this respect the disclosure of DE 196 00 301 and EP 0 874 851B1 is incorporated into the present invention by reference. Hence, if inthe scope of the present application a “carbon suboxide derivative” ismentioned, this term comprises all of the chemical compounds describedin DE 196 00 301. The preparation and characterization of the carbonsuboxide derivatives is also described in DE 196 00 301 and EP 0 874 851B1.

Thus the present invention especially relates also to combinedpreparations of at least one of the compounds according to the inventionand of at least one carbon suboxide derivative as described previouslyas well as in DE 196 00 301, EP 0 874 851 B1 and Kerek et al., Biochim.Biophys. Acta 1567, 213-220 (2002).

The present invention also comprises the cysteine containing peptidesaccording to the invention, the mixtures of these cysteine containingpeptides, the functional derivatives of these peptides and/or thepharmaceutically acceptable salts of these cysteine containing peptidesfor the preparation of a pharmaceutical composition for the treatment ofdiseases, especially for the treatment of diseases caused by pathogens.

In this context the preparation of a pharmaceutical composition for theprophylaxis and/or treatment of cancer provides special advantages, forexample in the case of choroidal melanoma, acute leukaemia, acousticneurinoma, ampullary carcinoma, anal carcinoma, astrocytoma, basal cellcarcinoma, pancreatic cancer, bladder cancer, bronchial carcinoma,breast cancer, Burkitt's lymphoma, corpus cancer, CUP-syndrome,colorectal cancer, small intestine cancer, small intestinal tumors,ovarian cancer, endometrial carcinoma, ependymoma, epithelial cancertypes, Ewing's tumors, gastrointestinal tumors, gallbladder cancer,uterine cancer, cervical cancer, glioblastomas, gynecologic tumors,throat, nose and ear tumors, hematologic neoplasias, hairy cellleukemia, urethral cancer, skin cancer, brain tumors (gliomas), brainmetastases, testicle cancer, lymph node cancer(Hodgkin's/Non-Hodgkin's), hypophysis tumor, carcinoids, Kaposi'ssarcoma, laryngeal cancer, germ cell tumor, bone cancer, colorectalcarcinoma, head and neck tumors, colon carcinoma, craniopharyngiomas,oral cancer (cancer in the mouth area and on lips), liver cancer, livermetastases, leukemia, eyelid tumor, lung cancer, lymphomas, stomachcancer, malignant melanoma, breast carcinoma, rectal cancer,medulloblastomas, melanoma, meningiomas, Hodgkin's disease, mycosisfungoides, nasal cancer, neurinoma, kidney cancer, non-Hodgkin'slymphomas, oligodendroglioma, esophageal carcinoma, osteosarcomas,ovarial carcinoma, pancreatic carcinoma, penile cancer, plasmocytoma,prostate cancer, pharyngeal cancer, rectal carcinoma, retinoblastoma,vaginal cancer, thyroid carcinoma, Schneeberger disease, esophagealcancer, spinal glioma, T-cell lymphoma (mycosis fungoides), thymoma,tube carcinoma, eye tumors, urethral cancer, urologic tumors, urothelialcarcinoma, vulva cancer, wart appearance, soft tissue tumors, Wilm'stumor, cervical carcinoma and tongue cancer.

Preferably the concerned cancer is chosen from the group comprisingbladder cancer, breast cancer, cancer of the central nervous system,colon cancer, stomach cancer, lung cancer, skin cancer, head and neckcancer, ovarian cancer, cervical cancer, glioblastomas, prostate cancer,testicular cancer, leukemia, liver cancer, kidney cancer and epithelialcancer types.

Also subject of the invention are further combined preparations of atleast one of the afore-mentioned compounds according to the inventiontogether with a cytostatic. Considered as cytostatics are alkylatingagents, antibiotics with cytostatic properties, antimetabolic agents,alkaloids, podophyllotoxins, platinum containing compounds, taxanes,cytostatic active agents and monoclonal antibodies. Examples for thesecompound classes are for example cyclophosphamide, ifosfamide,trofosfamide, temozolomide, chlorambucil, melphalan, busulfan,treosulfan, thiotepa, estramustine, nimustine, carmustine, lomustine,dacarbazine, procarbazine, adriamycin (doxorubicin), epirubicin(4-epi-adriamycin), idarubicin, actinomycin D, daunorubicin, bleomycin,dactinomycin, mitomycin C, mitoxantrone, methotrexate, 5-fluorouracil,capecitabine, cytosine arabinoside, thioguanine, mercaptopurine,fludarabine, cladribine, gemcitabine, vincristine, vinblastine,vindesine, etoposide, teniposide, cisplatin, carboplatin, oxaliplatin,paclitaxel, docetaxel, hydroxycarbamide (hydroxyurea), imatinib,miltefosine, amsacrine, topotecan (topoisomerase-I inhibitor),pentostatin, bexarotene, tretinoin, asparaginase, trastuzumab(Herceptin®), alemtuzumab (MabCampath®), rituximab (MabThera®).

The present invention also comprises pharmaceutics, which contain one ormore cysteine containing peptides according to invention and/orfunctional derivatives of these peptides. Especially preferred arepharmaceutics, which contain in addition at least one carbon suboxidederivative and/or one cytostatic and cytotoxic compound, respectively.

The cysteine containing peptides according to the invention can be usedboth as defense agents against pathogens and pharmaceutical activeagents for the control of the infections and diseases caused by thepathogens. Especially possible is the use against diseases which arecharacterized by a chronic deficient immunoregulation, such as forexample autoimmune diseases, cancer or AIDS. The cysteine containingpeptides according to the invention can be utilized separately as singlesubstances, but also as a mixture of several peptides or together withother already known active agents and substrate materials.

The combined preparations according to the invention as well as thepharmaceutical compositions according to the invention, which contain atleast one peptide according to the invention, are produced in a knownway using common solid or liquid substrates or diluents and commonlyused pharmaceutical adjuvants according to the intended type ofapplication with a suitable dosage. The preferred pharmaceuticalcompositions or preparations consist in a form which is suitable fororal administration or for inhalation. Such forms are for exampletablets, film tablets, layer tablets, coated tablets, capsules, microcapsules, pills, granulates, powders, solutions, dispersions,suspensions, suppositories, emulsions, dispersions, gels, ointments,syrup or depot forms or inhalation solutions and inhalation powders,respectively. In addition the pharmaceutical compositions according tothe invention comprise formulations such as layered tablets for thecontrolled and/or continuous release of the active agent as well asmicro encapsulations as special application forms.

Such pharmaceutical compositions are inter alia suitable for theinhalation or the intravenous, intraperitoneal, intramuscular,subcutaneous, mucocutaneous, oral, rectal, transdermal, topical,intradermal, intragastrical, intracutaneous, intravaginal, intranasal,intrabuccal, percutaneous or sublingual application. Especiallyadvantageous forms of application are oral adminstration, injection aswell as inhalation.

Appropriate tablets can be obtained, for example, by mixing theapplicable compound according to the invention and/or its salt withknown adjuvants, for example inert diluents such as dextrose, sugar,sorbitol, mannite, polyvinylpyrrolidone, disintegrating agents such ascorn starch or alginic acid, binding agents such as starch or gelatin,lubricants such as magnesium stearate or talcum and/or agents forproducing a depot effect such as carboxyl polymethylene, carboxymethylcellulose, cellulose acetate phthalate or polyvinyl acetate. The tabletsmay also consist of multiple layers.

Accordingly, pills can be produced by coating the cores produced in ananalogous way as the tablets with agents, that are typically used inpill coatings, for example polyvinylpyrrolidone or shellac, gum arabic,talcum, titanium dioxide or sugar. The pill coating may also consist ofmultiple layers, whereas the adjuvants mentioned above for tablets canbe used as well.

Solutions or suspensions with the active agent applicable according tothe invention may further contain taste enhancing agents such assaccharin, cyclamate or sugar as well as aromatizers such as vanillin ororange extract. They may further contain suspending adjuvants such assodium carboxymethyl cellulose or preservatives such asp-hydroxybenzoates. Capsules containing active agents can be produced,for example, by mixing the active agent with an inert substrate such aslactose or sorbitol, and encapsulating it in gelatin capsules.

Suitable suppositories can be produced, for example, by mixing with thesubstrates such as neutral fats or polyethylene glycol and derivativesthereof, respectively.

Such compositions are inter alia suitable for the inhalation or theintravenous, intraperitoneal, intramuscular, subcutaneous,mucocutaneous, oral, rectal, transdermal, topical, intradermal,intragastrical, intracutaneous, intravaginal, intranasal, intrabuccal,percutaneous or sublingual administration.

As pharmacologically acceptable substrates for example lactose, starch,sorbitol, sucrose, cellulose, magnesium stearate, dicalcium phosphate,calcium sulfate, talcum, mannite, ethyl alcohol and the like can beutilized. Powders as well as tablets can consist of 5 to 95% of such asubstrate.

As binders can be used in addition starch, gelatin, natural sugars,natural as well as synthetic gums such as for example acacia gum or guargum, sodium alginate, carboxymethyl cellulose, polyethylene glycol andwaxes. As lubricants can serve boric acid, sodium benzoate, sodiumacetate, sodium chloride, and the like.

Further disintegrating agents, coloring agents, flavoring agents and/orbinders can be added to the pharmaceutical compositions.

Liquid compositions comprise solutions, suspensions, sprays andemulsions. For example injection solutions based on water or onwater-propylene glycol for parenteral injections.

For the preparation of suppositories preferably low melting point waxes,fatty acid esters and glycerides are utilized.

Capsules are prepared for example from methyl cellulose, polyvinylalcohols or denatured gelatin or starch.

As disintegrating agents can be used starch, sodium carboxymethylstarch, natural and synthetic gums such as for example carob flour,karaya, guar, tragacanth and agar, as well as cellulose derivatives suchas methyl cellulose, sodium carboxymethyl cellulose, microcrystallinecellulose as well as alginates, clays and bentonites. These constituentscan be utilized in amounts from 2 to 30% by weight.

As binders can be added sugars, starch from corn, rice or potatoes,natural gums such as acacia gum, gelatin, tragacanth, alginic acid,sodium alginate, ammonium calcium alginate, methyl cellulose, sodiumcarboxymethyl cellulose, hydroxypropyl methyl cellulose,polyvinylpyrrolidone, as well as inorganic compounds such as magnesiumaluminum silicate. The binders can be added in amounts from 1 to 30% byweight.

As lubricants can be utilized stearates such as magnesium stearate,calcium stearate, potassium stearate, stearic acid, high melting pointwaxes, as well as water soluble lubricants such as sodium chloride,sodium benzoate, sodium acetate, sodium oleate, polyethylene glycol andamino acids such as leucine. Such lubricants can be used in amounts from0.05 to 15% by weight.

A preferred application of the cysteine containing peptides according tothe invention is in the support of the defense of biological organisms,especially plants, against bacterial, fungal, viral or other pathogens.The application can be carried out by administering the peptide into thenutrients/fluids incorporated by the plant or by applying the solutioncontaining the peptide onto the surface of the plant leafs.

The present invention also comprises the genetic integration of genesequences, which express a peptide according to the invention, into thegenome of an organism menaced by a disease, preferably into the genomeof plants. The increased resistance of the novel organism created bythis genetic alteration offers a clearly more gentle protection forhumans and the environment against pathogens than the chemicalpesticides known up to know.

The peptides according to the invention can be utilized in a similar wayfor the preventive fortification of the defense of other, especiallyanimal organisms, against pathogen infections of different types. Bythis way of application insects, nematodes and others carrying pathogensare successfully antagonized. Thereby the transfer and release,respectively, of the pathogens is prevented very efficiently and thistakes place before they can exert their pathogenic effect.

Another application of the peptides according to the invention iscarried out by application to a biologic, especially animal organism,already infected with a pathogen. Thereby the damaging consequences ofbacterial, viral or other infections are neutralized or at leastminimized. For this purpose the peptide or the mixture containing thepeptide is utilized in that it causes together with the organisms owndefenses a clearly more effective control of the pathogen.

The peptides according to the invention result in a significantreduction of the expression of some pro-inflammatory cytokines in humansand animals, especially of IL-2, IL-3, IL-4 and γ-IFN. Thus the peptidesaccording the to invention are capable of significantly reducing theauto-aggressive processes targeted against self tissue, especially inthe case of autoimmune diseases

By stimulation of inhibitory cytokines, especially of TGF beta, thepeptides according to the invention are capable of decreasing theactivity of the primary human immune cells, which are pathologicallyover-activated in the case of autoimmune diseases. Especially promisingexperimental results were obtained in the case of topical application ofthe peptides according to invention in the case of skin diseases withautoimmune character, especially in the treatment of psoriasis.

By stimulating the production of the suppressively acting andpotentially regulatory cytokine IL-10 the normal equilibrium between thepro- and anti-inflammatory cytokines, which is out of control in thecase of autoimmune diseases and other chronic diseases, is reestablishedby the peptides according to the invention.

The peptides according to the invention exert an efficient and partiallyselective inhibition on the propagation of malignant cancer cells. Thiscould be confirmed by cell culture experiments.

The present invention also comprises pharmaceutical compositions andpharmaceutics, which contain as the effective portion a peptideaccording to the invention or a mixture of peptides according to theinvention. The present invention further comprises pharmaceuticalcompositions and pharmaceutics, which contain a peptide according to theinvention or a mixture of peptides according to the invention togetherwith at least one already known active agent and/or together withpharmaceutically acceptable and suitable compounds and substrates.

The use of a peptide according to the invention together with the carbonsuboxide derivatives referred to as MCS results in a clearly improvedbioavailability and immunoregulatory effectiveness of the peptide,especially in the case of oncological applications (see examples).

For increasing the selectivity in the case of the control of malignanttumor cells the peptides according to the invention can be coupled withtumor-specific antigens. According to a preferred embodiment the peptidewith the sequence HT-C1 is bound to the antibody, which was generatedagainst human prostate carcinoma cells and isolated by affinitychromatography. The bonding is carried out by means of establishedcoupling reagents such as carbodiimides, preferably with the watersoluble 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDAC).

Compared to the use of single peptides according to the invention theuse of a mixture of the peptides according to the invention offers clearadvantages. In comparison to the presence of a single agent a mixture ofseveral active agents hampers the formation of pathogen resistance. Thesame advantages result in the case of both the biological defensethrough the transgenic expression of the peptide mixture and the controlof disease through the administration of the peptide to the alreadydiseased organism.

Thus the peptides according to the invention can be utilized in theabove described ways as an active agent. In addition to the use of thesingle peptides according to the invention the peptides according to theinvention are used as mixture of several peptides as well as togetherwith other substances, especially together with carbon suboxidederivatives. To be considered as forms of administration are injections,a spray and a topical application.

The peptides according to the invention are used in the case of all ofthe application types in a 0.0001 to 10% solution, especially in a 0.01to 1% solution, especially preferred in an about 0.2% solution.Especially preferred is the use of an aqueous peptide mixture. Thetreatment can be carried out for example by the daily administration of3×10 ml of the aqueous peptide solution.

The peptides according to the invention can be administered to thehumans, animals or plants to be treated as a pure compound or as apharmaceutical composition, wherein they are administered in atherapeutically effective dosage in a mixture with substrates ordiluents. Such therapeutically effective dose rates can be administeredseparately or in connection with other therapeutic compounds. In thecase of the use for the treatment of cancer in this context come intoconsideration antiproliferative and antiangiogenic agents such ascytostatic or cytotoxic anti-tumor agents such as 5FU, cisplatin or alsoprotein kinase inhibitors such as the Flk-1/KDR inhibitor CGP 79787,compounds of the class indolocarbazoles such as Gö7612, compounds of theclass bisindolymaleimides such as LY 333531, GF109203x, Ro 32-0432, Ro31-8220, compounds of the class balanol derivatives such as SPC 100840,compounds of the class antisense oligodeoxynucleotides such as CGP64128A and VEGF antisense oligonucleotide, compounds of the class of thealkyl lysophospholipids such as ET-18-OCH3, inhibitors of growth factorreceptor activation such as anti-HER2/neu antibody, trastuzumab(Herceptin), inhibitors of growth factor receptor kinase activity suchas compounds of the class phenylamino chinazolines such as PQ 153035, ZD1839 and CP-358774, compounds of the class substituted pyrimidinescomprising pyrido-, pyrrolo-, pyrazolo-, pyrimido- and phenylaminopyrimidines such as PD 158780, PD 166285, CGP 59326, CGP 60261 and CGP62706, compounds of the class tyrphostines AG1478, RG 13022 and AG 825,compounds of the class lavendustins such as lavendustin A, compounds ofthe class dianilinophthalimides such as CGP54698, as well as compoundschosen from the group comprising inhibitors of MAPKKK, inhibitors ofMAPKK, inhibitors of MAPK such as PD098059, U0126 or SB203580,interferon alpha, recombinant factor 4 for blood platelets, angiostatinor the poly-anionic compound suramin. The present invention alsocomprises the combination of the peptides according to the inventionwith one of the above indicated compounds.

Techniques for the formulation and administration of the peptidesaccording to the invention can be found in “Remington's PharmaceuticalSciences” Mack Publishing Co., Easton Pa. A composition, which comprisesone of the peptides according to the invention, can be present in theform of a solution of the peptide according to the invention, in aliquid pharmaceutical substrate or in any other formulation such astablets, pills, coated pills, capsules, gel, syrup, slurry, suspensionand the like.

SHORT DESCRIPTION OF THE DRAWINGS

The invention shall be explained in more detail in the following on thebasis of the embodiments in the context with the figures.

FIG. 1 shows the structure of the hellethionin HT-D;

FIG. 2 shows a HPLC diagram of a hellethionin mixture;

FIG. 3 shows the effect of different concentrations of the hellethioninHT-C on breast cancer cells of the type MCF-7.

WAYS OF REALIZING THE INVENTION Example 1 Extraction of a PeptideMixture

About 10 kg of roughly milled root or rootstock of Helleborus niger(Ranunculaceae family) are treated for 6 hours with 50 l of a TBM/hexanemixture (1:1). The defatted and air dried plant material is extractedtwo times with 60 l of EtOH 50% for about 24 h under slight stirring atroom temperature. The combined alcoholic-aqueous solutions are reducedin the vacuum rotary evaporator at 70° C. until dryness. The dry residueis treated three times with 3 l of 0.05 N hydrochloric acid, theresulting aqueous emulsions are combined and extracted successively with10 l each of hexane, chloroform and TBM. The aqueous phase is reducedunder vacuum to about 10 l of volume and the solution is treated withabout 200 g of active carbon (2 hours). The filtrate is reduced undervacuum up to 1.0 l. The aqueous concentrate is adjusted by means of a 1N HCl solution to pH value 1.2 and poured into a tenfold volume ofcooled (10° C.) acetone under strong stirring. The white precipitatethat is formed is separated from the supernatant by centrifugation anddried under vacuum. The dry precipitate is subsequently dissolved in aminimal amount of water and poured into an about tenfold volume ofcooled acetone under stirring. This precipitating procedure is repeatedonce again, the resulting mixture of the cysteine containing peptides isdissolved in water and lyophilized.

The mixture of the peptides according to the invention is characterizedby HPLC (FIG. 2). A Nucleosil 100-7, C-18 column (Macherey-Nagel Düren)with 250 mm length and 21 mm ID was used. At a flow rate of 3 ml/min andin a linear elution gradient of 20-50% of acetonitrile in 25 min thepeptides according to invention elute as follows: Hellethionin-A 14.4min. Hellethionin-B1 to hellethionin-B6 16.1 min. Hellethionin-C 16.9min. Hellethionin-D 18.3 min. Hellethionin-E1, hellethionin-E2 20.1 min.

Example 2 Extraction of the Pure Peptide HT-D

About 1 kg of milled roots of the plant Helleborus purpurascens areextracted for 4 hours with 10 l of diluted acetic acid (5% in water) at40° C. The filtered solution is reduced under vacuum up to 1 l.Afterwards about 400 g of ammonium sulphate is dissolved in thesolution. The precipitate that is formed is separated by centrifugation,dissolved in a minimal amount of water and then added to a mixture of3.6 l of acetone and 1.4 l of ethanol under strong stirring. Theformation of the precipitate in the acetone/ethanol mixture is repeatedtwo times and the resulting raw peptide mixture is dissolved in waterand lyophilized.

Afterwards 5 g of the lyophilized raw mixture of the peptide from thespecies Helleborus is dissolved in 100 ml of 20% acetonitrile/water with0.1% content of trifluoroacetic acid and separated into individualcomponents by means of the preparative high pressure liquidchromatography (HPLC). For this purpose the sample solution is added inaliquot amounts on a Nucleosil 100-7, C-18 column (Macherey-Nagel Düren)with 250 mm length and 21 mm ID (internal diameter). The separation iscarried out at a flow rate of 3 ml/min by means of a linear elutiongradient of 20-50% acetonitrile in 30 minutes. The individual purepeptides are collected by means of a sample collector of the typeFRAC-100 from Pharmacia-Biotech. The pure peptide HT-D is collected inthe retention time interval of 17.9-18.7 min.

The testing of the purity of the isolated peptide hellethionin-D iscarried out by analytic HPLC by means of a “Luna-CN” column fromPhenomenex (Offenbach) with 200 mm length and 4 mm diameter using alinear gradient of 5-85% of acetonitrile in 40 minutes.

The ¹H NMR spectrum of the pure hellethionin-D in water was correlatedwith the amino acid sequence determined through the Edman decompositionand the individual signals were assigned by means of NOESY- andTOCSY-spectra. Table 1 illustrates the individual resonance signals forHT-D obtained through this correlation. TABLE 1 ¹H NMR signalassignments for hellethionin-D Amino H—C H—C H—C Position acid H—N alphaH—C beta gamma delta others 1 Lys 3.72 1.36-1.47 0.83 1.1-1.03 HE: 2.64HZ: 7.17 2 Ser 8.52 4.65 3.41-2.96 3 Cys 8.45 4.6 4.22-1.93 4 Cys 9.474.9 2.60-2.14 5 Arg 7.52 3.51 1.65 1.35-1.30 2.93-2.62 H-E: 7.00 6 Asn6.82 4.46 3.01 7.51-6.45 7 Thr 8.3 3.58 3.79 0.94 8 Leu 7.65 3.771.38-1.29 0.57-0.61 9 Ala 8.02 4.07 1.52 10 Arg 7.68 4.16 1.92-1.36 1.593.06-3.30 HE: 8.07; HH: 6.10 11 Asn 8.16 4.21 2.64-2.60 6.7-7.29 12 Cys8.15 3.89 3.58-2.72 13 Tyr 8.74 3.36 3.06-2.89 HE: 6.40 14 Asn 8.55 42.62-2.52 7.6  15 Ala 7.63 3.93 1.19 16 Cys 7.98 3.8 2.83-2.78 17 Arg8.26 3.79 1.48-1.37 0.88-0.62 2.72-2.15 HE: 6.72; HH: 6.8-6.3 18 Phe 84.01 3.07-2.94 HD; HE; HZ: 7.01-6.91 19 Thr 7.24 3.94 4.27 1.06 20 Gly7.24 4.04-3.30 21 Gly 7.88 3.73-3.12 22 Ser 8.45 4.03 3.90-3.74 23 Gln9.01 3.76 1.84-1.73 2.20-1.95 1.95 HE: 7.1-6.58 24 Pr 4.05 1.99-1.561.78-1.65 3.54-3.39 25 Thr 7.1 3.55 3.8  0.84 26 Cys 8.4 4.28 2.29-2.1627 Gly 8.42 3.83-3.45 28 Ile 7.44 3.66 1.67 1.41-1.01 0.56 29 Leu 7.813.81 1.54-1.45 1.28 0.57 30 Cys 7.4 4.51 3.54-2.65 31 Asp 7.7 4.273.36-2.62 32 Cys 8.84 5.24 2.64-1.79 33 Ile 8.64 4.25 1.4  0.57-0.7−0.01  34 His 8.52 4.82 2.73-2.69 8.53 35 Val 7.96 4.44 1.99 0.44-0.3936 Thr 8.54 4.15 4.21 0.89 37 Thr 6.65 4.17 4.31 0.91 38 Thr 8.22 3.824.02 0.93 39 Thr 6.78 4.16 3.79 0.82 40 Cys 8.57 4.59 3.55-2.14 41 Pro4.29 1.98-1.84 1.5  3.51-3.30 42 Ser 8.55 3.94 3.71-3.59 43 Ser 7.623.87 3.76-3.65 44 His 7.41 4.37 2.21-2.01 6.72 HE: 8.44 45 Pro 4 1.661.66 3.42-3.07 46 Ser 7.78 4.04 3.42-2.56

Example 3 Effect of the Peptide HT-A on the Cytokine Production ofPrimary Immune Cells

The effect of the peptide HT-A on the cytokine production of primaryhuman immune cells was determined through the measurement of theconcentration of cytokines in lymphocyte cultures obtained from humanblood. Dosages of 4-200 μg/ml of the hellethionins were used, which wereobtained by adding the calculated amounts in the form of a stocksolution. The concentration of individual cytokines in the treated andcontrol samples is determined by means of the commercially available“Quantikine” ELISA plates from R&D Biosystems, Minneapolis, USA. Theresults were compared with control samples, i.e. with the lymphocyteculture without addition of the peptide.

In the case of the lymphocyte cultures (4 million cells per ml) treatedwith 4 μg and 200 μg of the peptide HT-A, respectively, the followingcytokine (and cytokine receptor) concentrations [μg/ml] were obtained incomparison with the control sample (without peptide): Control HT-A HT-ACytokine/peptide [without peptide] [4 μg/ml] [200 μg/ml] inhibited IL-2[pg/ml] 9.390 1.940 1.855 IL-3 [pg/ml] 41 26 8 IL-4 [pg/ml] 34 19 12γ-IFN [pg/ml] 11.605 7.454 7.504 IL-6R [pg/ml] 171 100 65 stimulatedIL-10 69 288 12 IL-2R 44 30 67 TGF-β2 9 105 45 not influenced IL-6 1.0951.100 1.130 IL-1RA 2.170 2.204 2.467 TNF-α 2.105 1.860 2.085

These data show, that the peptide HT-A is inhibiting the expression ofseveral pro-inflammatory cytokines such as for example IL-2, IL-3, IL-4and γ-IFN. By suppressing the production of these cytokines the use ofthe peptide results in the desired reduction of the harmfulauto-aggressive processes of autoimmune diseases.

Furthermore the peptide HT-A shows a stimulation of the cytokines actingsuppressively such as IL-10 and TGF-β12. Interestingly the stimulationof these cytokines in the case of a low peptide concentration (4 μg/ml)is clearly more intense than in the case of a higher dosage (200 μg/ml).It is assumed, that in the case of a higher peptide concentration thenon-specific cellular toxicity of the peptide overlaps the stimulationobserved in the case of lower dose rates.

The tested peptide HT-A shows no significant influence on the productionof some cytokines such as IL-6 or TNF-α and on the expression of theIL-1 receptor.

Example 4 Effect of the Peptide HT-C on the Proliferation of HumanCancer Cells

The investigations were carried out with the breast cancer cell cultureline MCF-7. A cell culture line from non differentiated breastepithelial cells of the type MCF-10A served as a comparison. Thecorresponding cells (about 10⁵/ml of sample) were initially stimulatedin the standard DMEM culturing medium. After 24 hours the peptide HT-Cwas added and concentrations between 0.2 and 400 μg/ml were adjusted inthis way. The examination of the alterations caused by the peptide wascarried out at intervals of 24 hours during a time period of up to 6days. The peptide HT-C already causes at low concentrations, namelyalready from 2 μg/ml, a very clear inhibition of the propagation of theMFC-7 cells (FIG. 3).

The peptide HT-C had a significantly minor inhibitory effect on thepropagation of the non malignant epithelial cells of the type MFC-10under the same conditions.

Example 5 Effect of the Peptide Mixtures on Tumour Development in Mice

In this study the effect of the peptides in mixture according to theinvention was investigated on tumour development in the female withWAZ-2T cells inoculated mice of the type BALB/c with a body weight of20-24 g. Initially the pathogenity of the cell strain was determinedthrough the inoculation of an increasing dose rate of 0-10⁶ tumour cellsin the right breast fat pad of the animals (12 mice/dose rate). Thispre-experiment showed, that the use of a dose rate of 2.5×10³ tumourcells resulted in a tumour incidence of 66.7%, i.e. a palpable tumourdeveloped in two of three mice. The diameter of the tumour was measureddaily and corrected with a value of 0.1 cm for the skin thickness of theanimal. The volume was calculated assuming a spherical shape of thetumour. The mice were sacrificed on day 120 or earlier, when the tumourhad reached a diameter of more than 1.8 cm. The 80 animals were groupedin four groups (I to IV) with 20 mice each. On day zero all of theanimals were inoculated with a suspension of 2.5×10³ tumour cells.

The animals of the group I (control group) received their normal foodwithout addition of the peptide mixture. In the case of animals reactingpositively The development of the tumour was measurable from the fourthweek after the inoculation. On day 50 after the inoculation a wellmeasurable tumour had developed in the case of a total of 14 animals ofthis group. Thus the tumour incidence on day 50 was 70%. The averagevolume of the tumour in the positive animals was determined as follows:Day 50 0.81 cm³ Day 60 1.23 cm³ Day 70 1.74 cm³

The animals of group II received from the first day of the inoculationdaily 0.4 μg of the peptide mixture according to the invention added totheir food. On day 50 after the inoculation a palpable tumour was wellmeasurable only in the case of 4 animals. Thus the tumour incidence onday 50 was 25%. The average volume of the tumour in the positive animalswas determined as follows: Day 50 0.32 cm³ Day 60 0.57 cm³ Day 70 0.71cm³

In the case of the animals of group III the treatment with the peptidemixture was started only from the 7^(th) day after the inoculation. Adaily dose rate of 0.4 μg of peptide mixture per animal was added to thefood. On day 50 after the inoculation a palpable tumour was wellidentifiable in the case of a total of 9 animals. Thus the tumourincidence on day 50 was 45%. The average volume of the tumour in thepositive animals was determined as follows: Day 50 0.48 cm³ Day 60 0.77cm³ Day 70 0.93 cm³

Thus the peptides according to invention cause a significant inhibitionof the malignant disease rate of the animals and a regression of thedevelopment of tumour.

Example 6 Effect of the Peptides on Murine Peritoneal Macrophages

Murine peritoneal macrophages extracted from CD1 mice were used. Aperitoneal flushing was carried out using 5 ml of PBS. After threewashing processes with RPMI 1640 the cells were put into a 96-wellculturing plate with a concentration of 1×10⁶ cells/ml (200 μl/well) in10% FCS-RPMI 1640 medium and kept for 24 hours. After the incubation thecells were washed three times with RPMI 1640 and resuspended in 10%FCS-RPMI 1640 medium (100 μl/well) in the absence (non stimulated) or inthe presence (stimulated) of different stimulants. After 24 hours ofincubation NO, IL-10 and TNF-α were measured in the culture supernatant.NO TNF-α IL-10 Survival Sample (μM) (pg/ml) (pg/ml) rate (%) Control 1.10 0 100 HT-C 5 μg/ml 0.7 244 6 80.3 HT-C 100 μg/ml 0.8 312 26 10.7 HT-D5 μg/ml 0.8 18 0 87.6 HT-D 100 μg/ml 1.3 0 0 42.8

The peptide HT-C shows a clearly stronger cytotoxic effect than HT-Dagainst the peritoneal macrophages from mice investigated herein. Thetested peptides do not induce NO in the murine peritoneal macrophages.

Example 7 Effect of the Individual Peptides and in Mixture with CarbonSuboxide Compound (MCS-18) on Cancer Cells of the Line COLO-205

Methods:

Cell line: Colon cancer cell line: Colo 205 (ATCC Nr. CCL-222),organism: homo sapiens, literature: Cancer Res., 38, 1345-1355, 1978.PNAS, 99, 10718-10723, 2002.

After adherence of the cells (incubation 24 h, cell amount 1×10⁴ cellsper batch), the cells were treated for 24 h with MCS and/or peptides(CZT, DZT) and afterwards the percentage of living cells was measured byMTT test.

Cell line: lung carcinoma cell line: A549 (ATCC No. CCL-185), organism:homo sapiens, literature: Giard D J, et al. J. Natl. Cancer Inst. 51:1417-1423, 1973.

After adherence of the cells (incubation 24 h, cell amount 5×10³ cellsper batch), the cells were treated for 72 h with MCS and/or peptides(CZT, DZT) and afterwards the percentage of living cells was measured byAlamarBlue test.

MTT-Test:

The MTT cell growth test was carried out according to Alley et. al. (M.C. Alley et al., Proc. Am. Assoc. Cancer Res., 27:389, 1986; M. C. Alleyet al., Cancer Res. 48:589-601, 1988). The finale concentration of3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide was 0.4mg/ml.

AlamarBlue™ cell growth test was carried out according to thespecifications of the manufacturer (Serotec, Oxford, England,www.serotc.com).

Result:

As shown in table 2 the growth of the Colo 205 cells is already stronglyinhibited after 24 hours at the concentration of 100 μg/ml in thepresence of the peptides CZT and DZT. The amount of the living cells isonly about 50%. When the substance MCS 18 is added in addition to thepeptide CZT, a still improved inhibiting effect of the peptide can beobserved. MCS 18 itself has at 100 μg/ml no inhibiting effect on thegrowth of the Colo 205 cells at all (table 3). Hence the combination ofCZT peptide and MCS can result in an increased anti tumour activity inthe case of colon cancer.

The peptides BZT, CZT and DZT were also able to inhibit the growth oflung carcinoma cell lines at a concentration of 100 μg/ml (see FIG. 4).Only about 50% of living cells could be determined after 72 h. TABLE 2Concentration Living Colo 205 cells Substance in [μg/ml] in [%] — — 100CZT peptide 50 92.15 CZT peptide 100 48.36 DZT peptide 50 92.15 DZTpeptide 100 58.66

TABLE 3 Concentration Living Colo 205 cells Substance in [μg/ml] in [%]— — 100 MCS 18 100 100 CZT peptide 50 92.15 CZT peptide 50 66.66 +MCS18100

1. Cysteine containing peptides of the structureXXCCXXXXXXXCXXXCXXXXXXQXXCXXXCXCXXXXXXXCXXXXXX or of the structureXXCCXXXXXXXCXXXCXXXXXXXXXCXXXCXCXXXXTXXCXXXXXX or of the structureXXCCXXXXXXXCXXXCXXXXXXXXXXCXXXCXCXXXXXXXXCXXXXXX, wherein X,independently of one another, represents any naturally occurring aminoacid.
 2. Cysteine containing peptides according to claim 1, wherein atposition 15 the amino acid G and/or at position 19 the amino acid Tand/or at position 23 the amino acid Q and/or at position 27 the aminoacid Q and/or at position 28 the amino acid R and/or at position 31 theamino acid D and/or at position 33 the amino acid I and/or at position34 the amino acid H and/or at position 35 the amino acid V and/or atposition 36 the amino acid T and/or at position 37 the amino acid Tand/or at position 38 the amino acid T and/or at position 43 the aminoacid S and/or at position 44 the amino acid H and/or at position 46 theamino acid S is located.
 3. Cysteine containing peptides according toclaim 1, namely KSCCRNTLGRNCYNGCRFTGGSQPTCGRLCDCIHVTTTTCPSSHPS(hellethionin-A), KSCCRNTLGRNCYNACRFTGGSQPTCGRLCDCIHVTTTTCPSSHPS(hellethionin-B1), KSCCRNTLARNCYNACRFTGGSQPTCGRLCDCIHVTTTTCPSSHPS(hellethionin-B2), KSCCRNTLGRNCYNACRLPGTPQPTCATLCDCIHVTTPTCPSSHPR(hellethionin-B3), KSCCRNTLARNCYNACRFTGTSQPYCARLCDCIHVTTPTCPSSHPR(hellethionin-B4), KSCCRNTLARNCYNACRFTGGSQPTCATLCDCIHVTTPTCPSSHPR(hellethionin-B5), KSCCRNTLARNCYNVCRFGGGSQAYCARFCDCIHVTTSTCPSSHPS(hellethionin-B6), KSCCRNTLGRNCYNACRLTGTSQATCATLCDCIHVTATTCRPPYPS(hellethionin-C), KSCCRNTLARNCYNACRFTGGSQPTCGILCDCIHVTTTTCPSSHPS(hellethionin-D), KSCCRNTLGRNCYAACRLTGLFSQEQCARLCDCITVTTPTPCPRTHPS(hellethionin-E1), KSCCRNTLGRNCYAACRLTGTFSQEQCARLCDCITVTTPTPCPRTHPS(hellethionin-E2).
 4. Nucleic acid sequence, which encodes a cysteinecontaining peptide compound according to claim
 1. 5. RNA sequence andanti-sense RNA according to claim
 4. 6. DNA sequence and anti-sense DNAaccording to claim
 4. 7. Ester derivatives, amide derivatives, halogenderivatives, methyl derivatives, salt derivatives, cyclic derivativesand derivatives with a modified backbone of the peptides according toclaim
 1. 8. DNA vector or DNA construct, which contains a DNA sequenceaccording to claim
 6. 9. Monoclonal antibodies targeted against anepitope of the cysteine containing peptides according to claim
 1. 10-32.(canceled)
 33. A method for the treatment or prophylaxis of a diseasecomprising administering to a patient in need thereof cysteinecontaining peptides according to claim 1, or functional derivatives ofthese peptides or mixtures thereof or pharmaceutically acceptable saltsof these compounds.
 34. The method of claim 33, wherein the disease tobe treated is caused by a pathogen, bacteria, fungi or viruses.
 35. Themethod of claim 33, wherein the disease to be treated is a disease ofhumans and animals, particularly of horses.
 36. The method of claim 33,wherein the disease to be treated is a disease caused by defectivebioregulation of the immune system or are accompanied by a defectivebioregulation of the immune system.
 37. The method of claim 33, whereinthe disease to be treated is an autoimmune disease, cancer or AIDS. 38.The method of claim 37, wherein the cancer is selected from the groupcomprising choroidal melanoma, acute leukaemia, acoustic neurinoma,ampullary carcinoma, anal carcinoma, astrocytoma, basal cell carcinoma,pancreatic cancer, bladder cancer, bronchial carcinoma, breast cancer,Burkitt's lymphoma, corpus cancer, CUP-syndrome, colorectal cancer,small intestine cancer, small intestinal tumors, ovarian cancer,endometrial carcinoma, ependymoma, epithelial cancer types, Ewing'stumors, gastrointestinal tumors, gallbladder cancer, uterine cancer,cervical cancer, glioblastomas, gynecologic tumors, throat, nose and eartumors, hematologic neoplasias, hairy cell leukemia, urethral cancer,skin cancer, brain tumors (gliomas), brain metastases, testicle cancer,lymph node cancer (Hodgkin's/Non-Hodgkin's), hypophysis tumor,carcinoids, Kaposi's sarcoma, laryngeal cancer, germ cell tumor, bonecancer, colorectal carcinoma, head and neck tumors, colon carcinoma,craniopharyngiomas, oral cancer (cancer in the mouth area and on lips),liver cancer, liver metastases, leukaemia, eyelid tumor, lung cancer,lymphomas, stomach cancer, malignant melanoma, breast carcinoma, rectalcancer, medulloblastomas, melanoma, meningiomas, Hodgkin's disease,mycosis fungoides, nasal cancer, neurinoma, kidney cancer, non-Hodgkin'slymphomas, oligodendroglioma, esophageal carcinoma, osteosarcomas,ovarial carcinoma, pancreatic carcinoma, penile cancer, plasmocytoma,prostate cancer, pharyngeal cancer, rectal carcinoma, retinoblastoma,vaginal cancer, thyroid carcinoma, Schneeberger disease, esophagealcancer, spinal glioma, T-cell lymphoma (mycosis fungoides), thymoma,tube carcinoma, eye tumors, urethral cancer, urologic tumors, urothelialcarcinoma, vulva cancer, wart appearance, soft tissue tumors, Wilm'stumor, cervical carcinoma and tongue cancer.
 39. Pharmaceuticalcomposition, comprising one or more cysteine containing peptidesaccording to claim 1 or functional derivatives of these peptides orpharmaceutically acceptable salts or mixtures of these compounds. 40.Pharmaceutical composition according to claim 39, further comprising atleast one carbon suboxide derivative.
 41. Pharmaceutical compositionaccording to claim 39, further comprising at least one cytostatically orcytotoxically active compound.
 42. Pharmaceutical composition accordingto claim 40, further comprising at least one cytostatically orcytotoxically active compound.
 43. Method for the extraction of thecysteine containing peptides according to claim 1 by extraction from theHelleborus plant species.
 44. Method according to claim 43, wherein adefatting of the plant material using non-polar solvents is carried outas first step of the method, particularly using tert.-butylmethylether.45. Method for the production of the cysteine containing peptidesaccording to claim 1 by gene technological methods.
 46. Method accordingto claim 45, wherein the thionine genes of a thionine producing corn arereplaced by the thionine genes of species of the plant Helleborus. 47.Method for the synthetic production of the cysteine containing peptidesaccording to claim 1 and of functional derivatives of these peptides bypeptide synthesis.